Lymphocyte numbers are regulated both by responses to conventional exogenous antigens and by stimulation by endogenous peptide/MHC complexes. This joint regulation permits individuals to maintain a broad repertoire of specificities, allowing responses against a vast array of foreign substances and, at the same time, providing a pattern of memory based on the immunization history of the individual. The study of the process of lymphocyte dynamics that underlies this regulation requires a multidisciplinary approach, aimed both at the molecular underpinnings of the processes through which lymphocytes survive and proliferate and a systemics approach to appreciate the overall mechanisms governing total numbers of lymphocytes of distinct phenotype and distinct specificity. Emphasis has been placed on three aspects of this problem: the dynamics of lymphocyte memory, the mechanisms underlying CD4 T cell depletion in HIV infection, and the process of homeostatic proliferation. During the past year, it has been shown that primary responses are highly dependent upon the number of precursor cells that can respond to antigenic challenge. Using both real time PCR and flow cytometric analysis to measure the response when TCR transgenic cells are transferred to intact recipients, it has been shown that responses of CD4 T cells are are not linear above 300 in the total populations of lymph node CD4 T cells. When limiting numbers of cells are transferred, the expansion factor on immunization of ~500. When larger numbers of precursors are present, the factor of expansion falls and may be as low as 20. The limitation in expansion does not result from a smaller fraction of cells responding but rather, at least in part, from early death of expanded cells when large numbers are present. Thus, the traditional approach of transferring millions of naive TCR transgenic cells to obtain information about primary immune responses cannot be expected to yield results that reflect physiologic responses. Our analysis of responses made by small numbers of transferred cells provides an approach to assess physiologic responses of naive cells. We are currently determining whether early death limits responses even within the physiologic numbers and whether the death is due to Fas-mediated killing as a result of high cell density or through competition for key cytokines or other growth/ survival factors. Analysis of the ?steady state? proliferation of memory CD4 T cells has yielded a striking and unanticipated finding. Naive CD4 T cells replicate very slowly; ~0.1% take up BrdU when mice receive a 6 hour ?pulse?. By contrast, among CD4 T cells with a memory phenotype, 3-5% take up BrdU in a 6 hour pulse. Furthermore, the cells that are dividing appear to replicate multiple times during a relatively short interval. When CD44bright CD4 T cells are labeled with CFSE, transferred to syngeneic mice and examined 7 days later, 10% to 15% of these cells have divided multiple times. This suggests either two populations of cells, one dividing and dying rapidly and one dying and dividing slowly or a process of TCR driven burst-like proliferation, perhaps representing ongoing responses either to exogenous antigens or internal peptides. The analysis of this process will give us important information on the ?global? behavior of CD4 T cells in vivo. We have continued the analysis of the factors regulating rapid proliferation of naive CD4 T cells on transfer into lymphopenic recipients. Our results indicate that it is not the number of resident memory cells that determines whether newly transferred cells may proliferate but rather the complexity of the repertoire of the resident memory cells. When naive cells are transferred into mice that have a simple repertoire of memory CD4 T cells, a substantial portion of the transferred cells undergoes rapid, TCR-dependent replication. These data imply that the immune system plays a premium on developing a population of memory CD4 T cells with a diverse repertoire and suggests that such cells play an important role, possibly in the initiation or mediation of early protective immune responses. Recent results indicate that when an animal has a small repertoire of CD4 T cells, and an equally small (actually smaller) repertoire of regulatory T cells, that animal is at very severe risk of developing a fatal pneumonia characterized by infiltration with alternatively activated macrophages and with numerous eosinophils. This pneumonia can be prevented by administering purified CD25+ CD4+ T cells to the Rag2-/- mouse one month prior to the transfer of limited numbers of CD4 T cells. This suggests that inadequate numbers of regulatory T cells or more likely the failure of repertoire-matching between regulatory and effector T cells is key to the development of thie "Th2 pneumonia". A detailed analysis of the cellular and molecular basis of the Th2 response underlying this effect is underway.